The study of proteomics encompasses the study of individual proteins and how these proteins function within a biochemical pathway. Proteomics also includes the study of protein interactions, including how they form the architecture that constitutes living cells.
In order to isolate the individual proteins for the complex mixtures and characterize the properties on the proteins, techniques such as affinity chromatography are employed. Affinity chromatography is a method for separating protein mixtures and based on a very specific biological interaction, for example, interactions between an antigen and an antibody, or an enzyme and a substrate. Affinity chromatography entails the ability to design a chromatography that reversibly binds the protein to a known subset of molecules.
Protein complexes are now routinely immunoisolated from cell lysates via an affinity tagged member of the complexes. Affinity chromatography has become suitable for any organism for which there is an affinity handle for at least one of its proteins. This technology is widely used because of the relative ease of incorporating a genomic tag by homologous recombination, and also to the commercially available TAP-tag (tandem affinity purification) collection of dual affinity-tagged proteins. The immunoisolation technique is an exceptionally powerful method for rapidly and efficiently extracting a protein complex from cell lysate under conditions that attempt to preserve in vivo protein interactions.
US Patent Publication 2007/0077600A1, discloses that a common problem with the TAP-tag methods is the co-enrichment of proteins that associate non-specifically with affinity-tagged proteins. The 2007/0077600A1 publication proposes a method of determining whether or not associations between a given protein and other proteins in a cell are specific.
In an article entitled, “Experimental Approaches to Protein-Protein Interactions,” by Mike P. Williamson and Michael J. Sutcliffe, it is disclosed that although there is an increase in demand for the high throughput analysis techniques like the TAP-tag method, these methods suffer from the problem that they depend on the formation of dilute solutions outside of the cell, and even though these methods employ mild interactions to isolate and purify individual proteins, weak interactions between proteins are lost. Generally, the intercellular environment is crowded, with protein occupying up to about 40 percent of the total fluid volume of the cell. Thus, within the cell, the protein-protein interactions will be stronger than they would be in dilute solution. Therefore, methods for purification outside the cell implies that weaker interactions will be lost.
Protein-protein interactions are typically identified and characterized by means of low-throughput biophysical methods. These biophysical methods may include nuclear magnetic resonance (NMR), crystallography, spectroscopic methods, chromatographic, mass spectroscopic, and calorimetric methods. Current methods are either low resolution and somewhat unreliable, or high resolution and low throughput.
There is also a need to develop technologies for analysis of the proteome that allow scaling up to industrial levels with the features of an industrial process: high accuracy, reproducibility and flexibility in that the process is high-throughput, automatable and cost-effective. There is a need to develop technologies that permit probing and identification of proteins in their native conformation using automated protocols and systems. In particular, there is a need to develop strategies and technologies for identification and characterization of hydrophobic proteins under physiological conditions.
This information can be gathered slowly in serial, or rapidly in parallel. Moreover, when this information is gathered in parallel, it has emergent-value related to the pattern of the information that is generally not obtained, or not equivalently obtained, in conventional serial fashion. Prior attempts for analysis of the proteome by affinity chromatography methods using a multi-well format have experiences problems in the stability of the well structure and uneven flow or plugging in the filter media.
It is desired to have a stable multi-well filtration system with uniform distribution of fluids throughout the multi-well filter when using centrifugation methods or positive pressure methods to provide the collection of interacting proteins in an affinity chromatography process.